Modular tissue scaffolds

ABSTRACT

Provided are biocompatible and implantable scaffolds for treating a tissue defect, such as a bone gap. The scaffolds can have a modular design comprising a tissue scaffold rack designed to accommodate one or more modules. Also provided are methods for fabrication and use of such scaffolds.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Application Ser. No. 61/447,352 filed 28 Feb. 2011, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to tissue scaffolds. Morespecifically, degradable tissue scaffolds are provided having modulesfor filling a gap in a tissue, where a variable number of modules areinserted into the scaffold as needed to fill the gap.

BACKGROUND OF THE INVENTION

Extensive research has been devoted to the development of degradabletissue scaffolds to fill bone, cartilage or soft tissue defects(Hollister, 2009). The scaffolds that have been developed are generallycustom designed and prepared for a defect in a particular individual,are prepared in standardized sizes, or are initially flowable so thescaffold can be injected to fill the tissue gap. For defects that arevariable in size, for example a defect in a mandible or long bone due totumor resection or injury, the scaffolds must be custom designed to fitthe defect. This is an expensive, time consuming process that canpreclude the use of scaffolds in favor of more traditional approachessuch as the grafting of free flap autografts.

Modular orthopaedic implants that can be expanded have been described(see e.g., U.S. Pat. No. 7,481,841, describing a metal prosthesis thatmay be adjusted via a radio signal; U.S. Pat. No. 7,468,078, describinga modular hip prosthesis with different ball and stem; U.S. Pat. No.7,455,695, describing a femoral stem modular prosthesis withinterlocking nut; U.S. Pat. No. 7,453,263, describing a modular femoralhead and neck prosthesis; U.S. Pat. No. 7,309,361, describing a coupledmetallic tibia and femoral implant with resorbable lining; and U.S. Pat.No. 7,297,164, describing a modular knee prosthesis with tibial andfemoral components broken into medial and lateral sides. But suchmodular orthopaedic implants have generally been made from permanentmaterials, or at most a combination of a permanent material with adegradable liner (see e.g., U.S. Pat. No. 7,309,361). Furthermore,permanent materials of conventional modular implants do not provide forsurface release of biologic factors individually or separately from oneor more individual modules.

While permanent materials have a long history of clinical use, they alsohave significant drawbacks. Firstly, they are radioopaque, which makesevaluating the degree of healing post-operatively difficult. Secondly,there is a large difference between the elastic modulus of the metalimplant and that of the adjacent bone. This can cause stress shielding,which in turn can lead to complications including: implant/screwloosening, future instrumentation failure, device-related osteopenia,soft tissue dehiscence, and fracture. Finally, micromotion of the metaldevice can create wear debris that triggers an inflammatory response.More recently, devices have been composed from non-degradable polymers,most notably polyether-etherketone (PEEK). While these devices do havethe advantage of being radiolucent, the mismatch between the modulus ofthe material and the bone as well as the potential for wear debris stillexist.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofa tissue scaffold comprising a first module that is biocompatible anddegradable when implanted into a vertebrate, long bone, mandible orcranium, wherein the first module is designed to couple to a secondmodule that is biocompatible and degradable when implanted into amammal, and wherein the first module comprises an A connector designedto couple to a B connector present on the second module.

In another embodiment of a biocompatible system for filling a tissuegap, the system comprises a first tissue scaffold module that isbiocompatible and degradable when implanted into a vertebrate, whereinthe first tissue scaffold module is designed to couple to a secondtissue scaffold module that is biocompatible and degradable whenimplanted into the vertebrate, additional tissue scaffold modules asneeded to fill the tissue gap when joined to the first tissue scaffoldmodule, the second tissue scaffold module, or one of the additionaltissue scaffold modules, and a biocompatible rack designed toaccommodate the first tissue scaffold module, the second tissue scaffoldmodule and the additional tissue scaffold modules.

In another embodiment of a biocompatible system for filling a gap in along bone of a mammal, the system comprises at least one tissue scaffoldmodule that is degradable when implanted in the mammal, wherein eachmodule has an irregular disk shape having two flat sides and each modulecomprises a dovetail connector on each flat side, wherein the dovetailconnector from one module is designed to couple with a dovetailconnector from another module and wherein the circumference of theirregular disk shape is substantially in the form of an outline ofmissing tissue in the gap in the long bone, and a biocompatible rackcomprising a trough shaped portion having two side regions, a bottomregion, a proximal end and distal end, wherein the modules fit into thetrough shaped region by contacting the bottom region and substantiallyspanning the two side regions, wherein each module and the rack have aporous microstructure, are synthesized from polycaprolactone and aresubstantially coated with calcium-deficient carbonate-containinghydroxyapatite, and wherein the system comprises a sufficient number ofmodules to substantially fill the gap.

In another embodiment of a biocompatible system for filling a gap in amandible of a mammal, the system comprises at least one tissue scaffoldmodule that is degradable when implanted in the mammal, wherein eachmodule has an irregular disk shape having two flat sides and each modulecomprises a dovetail connector on each flat side, wherein the dovetailconnector from one module is designed to couple with a dovetailconnector from another module and wherein the circumference of theirregular disk shape is substantially in the form of an outline ofmissing tissue in the gap in the mandible, a biocompatible rackcomprising a trough shaped portion having two side regions, a bottomregion, a proximal end and distal end, wherein the modules fit into thetrough shaped region by contacting the bottom region and substantiallyspanning the two side regions, wherein the rack spans the mandible gapby the ends of the trough shaped region partially enveloping themandible, wherein each module and the rack have a porous microstructure,are synthesized from polycaprolactone and are substantially coated withcalcium-deficient carbonate-containing hydroxyapatite, and wherein thesystem comprises a sufficient number of modules to substantially fillthe gap.

In one or more embodiments the tissue scaffold further comprises asecond module, wherein the second module is joined to the first moduleby coupling the B connector of the second module to the A connector ofthe first module. In another embodiment the A connector is integral tothe first module and the B connector is integral to the second module.In some embodiments the A connector is identical to the B connector. Insome embodiments the A connector is not identical to the B connector. Insome embodiments the A connector and B connector are dovetailconnectors. In some embodiments the dovetail connectors are elliptical.In some embodiments the first module and second module comprise both anA connector and a B connector.

In some embodiments, the tissue scaffold further comprises a thirdmodule that is biocompatible and degradable, wherein the third modulecomprises both an A connector and a B connector and is joined to thefirst module or the second module by the third module A connector or Bconnector. In some embodiments the first module and second module eachhave an irregular disk shape having two flat sides, wherein the Aconnector is on one flat side and the B connector is on the other flatside, and wherein the circumference of the irregular disk shape issubstantially in the form of an outline of missing tissue of a tissuegap. In some embodiments the first module and second module each have anirregular disk shape having two flat sides, the first module comprisesan A connector on one flat side and an A connector on the other flatside, the second module comprises a B connector on one flat side and a Bconnector on the other flat side, and the circumference of the irregulardisk shape is substantially in the form of an outline of missing tissueof a tissue gap.

In some embodiments the first module and the second module comprise abioactive agent or a vertebrate cell, wherein the bioactive agent orvertebrate cell in the first module is different from the bioactiveagent or vertebrate cell in the second module. In some embodiments thefirst module and second module both have a porous microstructure. Insome embodiments the first module and second module are synthesized froma material independently selected from a degradable polymer and amixture of a degradable polymer and a bioceramic. In some embodimentsthe polymer is polycaprolactone, polylactide, polyglycolide,poly(lactide-glycolide), poly(propylene fumarate), poly(caprolactonefumarate), polyethylene glycol, poly(glycolide-co-caprolactone), ormixtures thereof. In some embodiments the polymer is polycaprolactone.

In some embodiments the first module and/or the second module furthercomprise a ridge designed to be melted by the application of energy,wherein the application of energy to the ridge fuses the first module tothe second module. In some embodiments the ridge is on the A connectorand/or on the B connector. In some embodiments the first modulecomprises an osteoconductive mineral coating on at least a portion ofthe module. In some embodiments the osteoconductive mineral coating ishydroxyapatite, calcium-deficient carbonate-containing hydroxyapatite,tricalcium phosphate, amorphous calcium phosphate, octacalciumphosphate, dicalcium phosphate, calcium phosphate, or a mixture thereof.In some embodiments the osteoconductive mineral coating iscalcium-deficient carbonate-containing hydroxyapatite.

In some embodiments the tissue scaffold comprises a bioactive agent. Insome embodiments the bioactive agent is with the first module. In someembodiments the bioactive agent is present in an amount that inducesossification. In some embodiments the bioactive agent is a bonemorphogenetic protein (BMP), demineralized bone matrix, a bone marrowaspirate, a transforming growth factor, a fibroblast growth factor, aninsulin-like growth factor, a platelet derived growth factor, a vascularendothelial growth factor, a growth and development factor-5, plateletrich plasma, or a mixture thereof. In some embodiments the bioactiveagent is BMP2 or BMP7.

In some embodiments the tissue scaffold comprises a vertebrate cell. Insome embodiments the vertebrate cell is a mammalian cell. In someembodiments the vertebrate cell is with the first module. In someembodiments the vertebrate cell is a stem cell. In some embodiments thestem cell is an embryonic stem cell. In some embodiments the stem cellis an adult stem cell. In some embodiments the stem cell is amesenchymal stem cell or an induced pluripotent stem cell.

In some embodiments the A connector is identical to the B connector, thefirst module has a porous microstructure, the first module issynthesized from polycaprolactone, and the first module is substantiallycoated with calcium-deficient carbonate-containing hydroxyapatite.

In some embodiments the tissue scaffold further comprises a tissuescaffold rack designed to accommodate the first module and the secondmodule, wherein the first module and second module are joined to thetissue scaffold rack. In some embodiments the tissue scaffold isdesigned to span a tissue gap in the vertebrate. In some embodiments therack is degradable when implanted into a mammal.

In some embodiments the rack has a porous microstructure, the rack issynthesized from polycaprolactone, and the rack is substantially coatedwith calcium-deficient carbonate-containing hydroxyapatite. In someembodiments the first module and/or the second module and/or the rackfurther comprise a ridge designed to be melted by the application ofenergy, wherein the application of energy to the ridge fuses the firstmodule and/or the second module to the rack, and/or the first module tothe second module. In some embodiments each module further comprises a Cconnector that can couple to a D connector and wherein the D connectoris on the rack. In some embodiments the rack comprises a plurality of Dconnectors that can couple to each of the modules of the tissue scaffoldthrough a C connector on each module. In some embodiments the firstmodule and/or the second module and/or the rack further comprise a ridgedesigned to be melted by the application of energy, wherein theapplication of energy to the ridge fuses the first module or the secondmodule to the rack, and wherein the ridge is on the C connector and/oron the D connector.

In some embodiments the rack comprises a trough shaped portion havingtwo side regions, a proximal end and distal end. In some embodiments therack further comprises a bottom region, wherein the modules fit into thetrough shaped region by contacting the bottom region and substantiallyspanning the two side regions. In some embodiments the rack furthercomprises D connectors in the bottom of the trough shaped region thatcouple to C connectors on the modules where the modules contact thebottom of the trough shaped region. In some embodiments the D connectoris a recess and the C connector is a protuberance on the module, whereinthe protuberance fits into the recess. In some embodiments the Dconnector is a protuberance and the C connector is a recess on themodule, wherein the protuberance fits into the recess.

In some embodiments the rack spans a bone gap in a mammal and themodules fill the gap. In some embodiments the bone gap is in a longbone. In some embodiments the rack spans the long bone gap by the endsof the trough shaped region partially enveloping the long bone. In someembodiments the bone is a mandible of a living mammal. In someembodiments the rack spans a gap in the body of a mandible by the endsof the trough shaped region partially enveloping the body of themandible. In some embodiments the rack comprises a bar which the modulesat least partially envelop. In some embodiments the rack is notdegradable. In some embodiments the rack is degradable.

In some embodiments the rack has a porous microstructure, the rack issynthesized from polycaprolactone, and the rack is substantially coatedwith calcium-deficient carbonate-containing hydroxyapatite.

In some embodiments each tissue scaffold module and/or the rack furthercomprises a ridge designed to be melted by the application of energy,wherein the application of energy to the ridge fuses at least one of themodules to at least another module and/or the rack. In some embodimentsthe dovetail connectors are elliptical.

In an embodiment of a method of filling a tissue gap, the methodcomprises inserting the tissue scaffold of an embodiment described aboveinto the tissue gap. In another embodiment the method further comprisesfusing the first module to the second module by placing a liquifiedbiocompatible polymer between the first module and the second modulesuch that the polymer hardens and fuses the first module to the secondmodule. In another embodiment the method further comprises applyingenergy to the ridge such that the ridge melts and fuses the first moduleto the second module.

In another embodiment, the method comprises partially enveloping the gapin the long bone with the rack such that the rack spans the gap,inserting a module into the trough shaped region of the rack such thatthe module spans the two top edges of the trough shaped region, andinserting additional modules into the trough shaped region as necessaryto fill the gap, wherein the modules couple with each other at thedovetail connector. In another embodiment the method further comprisesfusing the modules to each other and/or to the rack by placing aliquefied biocompatible polymer between each of the modules and/orbetween the modules and the rack such that the polymer hardens and fusesthe first module to the second module and/or the rack.

In another embodiment of the method each tissue scaffold module and/orthe rack further comprise a ridge designed to be melted by theapplication of energy, wherein energy is applied to the ridge such thatat least one of the modules is fused to at least another module and/orthe rack. In another embodiment of the method the tissue gap is in amandible of a mammal, and the method comprises partially enveloping thegap in the mandible with the rack such that the rack spans the gap,inserting a module into the trough shaped region of the rack such thatthe module spans the two top edges of the trough shaped region, andinserting additional modules into the trough shaped region as necessaryto fill the gap, wherein the modules couple with each other at thedovetail connector. In another embodiment the method further comprisesfusing the modules to each other and/or to the rack by placing aliquefied biocompatible polymer between each of the modules and/orbetween the modules and the rack such that the polymer hardens and fusesthe first module to the second module and/or the rack.

In another embodiment the tissue scaffold further comprises at least onepin, wherein the at least one pin is used to couple the tissue scaffoldto tissue.

In another embodiment the tissue is bone. In another embodiment the atleast one pin is made from a biodegradable and absorbable material. Inanother embodiment the at least one pin is coupled to the tissuescaffold and adjacent tissue. In another embodiment the coupling isaccomplished by at least one of sonically welding and applying energy tobond the at least one pin to the tissue scaffold and the adjacenttissue. In another embodiment the pin is synthesized from a materialindependently selected from a degradable polymer and a mixture of adegradable polymer and a bioceramic. In another embodiment the polymeris polycaprolactone, polylactide, polyglycolide,poly(lactide-glycolide), poly(propylene fumarate), poly(caprolactonefumarate), polyethylene glycol, poly(glycolide-co-caprolactone), ormixtures thereof. In another embodiment the polymer is polycaprolactone.

In another embodiment the pin comprises an osteoconductive mineralcoating on at least a portion of the pin. In another embodiment theosteoconductive mineral coating is hydroxyapatite, calcium-deficientcarbonate-containing hydroxyapatite, tricalcium phosphate, amorphouscalcium phosphate, octacalcium phosphate, dicalcium phosphate, calciumphosphate, or a mixture thereof. In another embodiment theosteoconductive mineral coating is calcium-deficientcarbonate-containing hydroxyapatite.

In another embodiment the pin comprises a bioactive agent. In anotherembodiment the bioactive agent is present in an amount that inducesossification. In another embodiment the bioactive agent is a bonemorphogenetic protein (BMP), demineralized bone matrix, a bone marrowaspirate, a transforming growth factor, a fibroblast growth factor, aninsulin-like growth factor, a platelet derived growth factor, a vascularendothelial growth factor, a growth and development factor-5, plateletrich plasma, or a mixture thereof.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

FIG. 1 is an image showing a sleeve design for a mandibular condylereconstruction. FIG. 1A shows a longitudinal view of sleeve highlightinginternal raised slots for module insertion. FIG. 1B shows a transverseview showing overall sleeve design.

FIG. 2 is an image showing a module design with slots for mating intosleeve fixation and dovetail joint for fitting each module together.

FIG. 3 is an image showing a sleeve module design applied to a tibialsegmental defect. The outer ends of the bone are the remaining tibia,the middle section represents the module region, and the pair of wrappedsections between the outer ends and middle represent a sleeve areaattached to the remaining bone. Modules can also be contained using acentral core.

FIG. 4 is an image showing a modular scaffold made from a degradablepolymer using a laser sintering techniques. Modules are placed in thescaffold in the upper portion of the image and laid out individually inthe lower portion of the image.

FIG. 5 is a series of photographs showing creation of a surgical defect,bioresorbable pin placement, and complete welded scaffold in an adultYorkshire pig model. FIG. 5A shows initial creation of a 3.5 cmmandibular segmental defect. FIG. 5B shows initial sizing of scaffoldsleeve with pins (rectangular highlight) located in drill holes. FIG. 5Cshows final scaffold implantation and welding of modules (upper middlerectangular highlight) and welding of pins (middle flanking rectangularhighlights).

FIG. 6 is a series of images showing bone fill and correct anatomicshape of an implanted scaffold at 6 months in vivo. FIG. 6A shows a 3Dreconstruction of a bone bridging original defect (defect margins shownby vertical lines). FIG. 6B shows bone growing in scaffold module withmodule pore structure (rectangular highlight). FIG. 6C shows bone filein a second pig defect.

FIG. 7 is a pair of images showing bioresorbable pin fixation after 6months in vivo. FIG. 7A shows dual pin tracts in mandible (tractsoutlined with four solid straight lines). FIG. 7B is an image two slicesaway (1.2 mm) from FIG. 7A showing bone formation (circular highlight)underneath lower pin tract (tracts outlined with two solid straightlines).

FIG. 8 shows a medial-lateral view of a modular scaffold design withmarrow space interfaces that mechanically stabilize the endoprosthesisin addition to pins that are placed through the marrow space interfaces.

FIG. 9 shows an anterior-posterior view of the modular scaffold designfrom FIG. 8 with associated pins.

FIG. 10 shows placement of a modular mandibular reconstruction scaffoldbased on the designs shown in FIGS. 8 and 9 within a mandibular defectin a pig mandible.

DETAILED DESCRIPTION OF THE INVENTION

Provided herewith are scaffolds 10 with modular components that can beused for tissue reconstruction, e.g., to fill defects of variable sizes.The scaffolds 10 comprise biocompatible or degradable tissue modules 30,a variable number of which can fill a defect of any size.

A scaffold 10 provided herein can be used to fill any type of tissuedefect, for example a soft tissue defect, a cartilage defect, or a bonedefect 45. A scaffold 10 described herein can be used for long bones orany other anatomic regions. For example, a scaffold 10 having a rack 20,in the form of two sleeves containing a central region, and one or moremodules 30 can be used to fill a defect in a long bone (see e.g., FIG.3). At least one advantage of a modular design is the ability to adjusta dimension (e.g., length) an implant by adding or removing a module.Such adjustment can be accomplished during a surgery or in an operatingroom, thus eliminating a need for manufacture of multiple implants eachof a different fixed size. Such a design also has advantages for cell,gene, protein and drug delivery using various coatings. These and otherfeatures are discussed further below.

A scaffold 10 described herein can have mechanical properties the sameor substantially similar to that of bone 40. Thus stress shielding canbe avoided. Because various embodiments of the implantable scaffold 10can be resorbed and replaced by native bone 40, problems associated withwear debris and long term foreign body reactions can be reduced. Ascaffold 10 or component can be fabricated from polymer materialsdescribed herein. Some polymers provide for a radiolucent implantablescaffold 10. A scaffold 10 or component described herein can include anosteoconductive coating that can bind to one or more optionalosteoinductive agents (e.g., a agent naturally occurring in the body ofa subject or an agent introduced peri-operatively). A scaffold 10 orcomponent described herein can be osteoinductive or provide for releaseof factors in a temporally or spatially controlled manor from one ormore components or modules 30.

Described herein is an implantable scaffold 10 that can include one ormore modular components, a tissue scaffold rack 20, or some combinationthereof. The rack 20 can interface with tissue, such as bone 40, near toa tissue defect. For example, the rack 20 can interface with bone 40surrounding or flanking a bone defect 45. The rack 20 can accommodateone or more scaffold modules 30. For example, one or more modules 30 canbe placed in or on a scaffold rack 20 such that the module(s) partiallyor substantially fill a tissue defect.

A scaffold 10 described herein can be implanted to correct a tissuedefect in bone 40 tissue. For example, a scaffold 10 can span a tissuedefect, such as a gap. A scaffold 10 can be implanted in a vertebratesubject, such as a mammal subject. For example, a scaffold 10 can spanand fill or substantially fill a bone gap in a mammal. As anotherexample, a scaffold 10 can span a bone gap (e.g., a long bone gap) withthe ends of the scaffold rack 20 partially or substantially envelopingthe bone 40 and one or modules 30 filling or substantially filled thegap. A scaffold 10 can be designed to correct a tissue defect in a bone40 mandible of a living mammal. For example, a scaffold 10 can span agap in the body of a mandible with the ends of the scaffold rack 20partially or substantially enveloping the body of the mandible and oneor modules 30 filling or substantially filled the gap in the mandible.

Design of Scaffold

Scaffold 10 design can be according to U.S. Pat. No. 7,174,282, whichprovides a non-limiting example of a design methodology for creatingbiomaterial scaffolds 10 with internal porous architectures that meetthe need for mechanical stiffness and strength and the need forconnected porosity for cell migration and tissue regeneration (see also,US Pat App Pub No. 2008/0195211, US Pat App Pub No. 2008/0215093 and USPat App Pub No. 2006/0276925). Design methods, such as those describedin US Pat App Pub No. 2003/0069718 can combine image-based design ofstructures with homogenization theory to compute effective physicalproperty dependence on material microstructure. Optimization techniquescan then be used to compute the optimal geometry. The final optimizedscaffold 10 geometry voxel topology can be combined with a voxel dataset describing the three dimensional anatomic scaffold 10 shape whichmay be obtained by imaging techniques such as magnetic resonance (MR)images or combined MR and computed tomography (CT) images. Densityvariations within the anatomic scaffold 10 voxel database can be used asa map to guide where different optimized scaffold 10 voxel topologiesare substituted. The final voxel representation of the anatomicallyshaped scaffold 10 with optimized interior architecture can then beconverted automatically by software into either a surface representationor wire frame representation for fabrication of the scaffold 10 by wayof solid free form fabrication or casting.

The methods described in US Pat App Pub No. 2006/0276925 also provide adesign methodology for creating biomaterial scaffolds 10 with internalporous architectures that can provide adequate mechanical stiffness andstrength for any scaffold, and the need for connected porosity for cellmigration and tissue regeneration. The methods of US Pat App Pub No.2006/0276925 can be used to generate a scaffold 10 with a designedperiodic microstructure that attains desired stability (displacements<0.9 mm), while maintaining compliance to avoid stress shielding and alarge porosity for biofactor delivery.

Using any of the methods described herein, once a scaffoldingimage-design dataset is created, it can be automatically converted intoa surface representation in, for example, .stl file format(stereolithography triangular facet data). This makes it possible tofabricate the scaffolding from any type of Solid Free-Form Fabrication(SFF) system using either direct or indirect methods. Direct SFF methodsinclude, but are not limited to: (1) Selective Laser Sintering (SLS);(2) Stereolithography (SLA); (3) Fused Deposition Modeling (FDM); (4) 3Dprinting (3DP), and (5) Selective Laser Melting (SLM). Conventionaldesign of the scaffolds 10 and newer design by degradation topologyoptimization can be exported to an EOS Formega P 100 machine (3DSystems, Valencia, Calif., USA) in .stl file format, and can be used toconstruct scaffolds 10 by SLS processing of e.g., ϵ-polycaprolactonepowder. This particular form of polycaprolactone has a melting point of60° C., a molecular weight in the range of 35,000 to 100,000 Daltons,and particle size distribution in the 25-100 pm range. However,nanoscale particle sizes can also be suitable in place of the microscaleparticle sizes. Scaffolds 10 are built layer-by-layer using a powderlayer thickness of, e.g., 100 pm. After SLS processing is completed, thescaffold 10 is allowed to cool inside the machine process chamber and isthen removed from the part bed. Excess powder surrounding the cages isbrushed off and the scaffolds 10 are finally cleaned by blowingcompressed air and physically removing unsintered powder from the cageinterstices by insertion of a 1 millimeter diameter wire.

In some embodiments, SLS parameters are divided into five maincategories—Contour 1, Contour 2, Edge 1, Edge 2, and Hatching.

Modules

Implantable scaffolds 10 described herein can include one or moremodules 30. A scaffold 10 can comprise modular inserts 30 with adesigned porosity that can be fitted within a rack 20 designed to fit aan anatomical region or a center core that runs between two anatomicalregion. For example, a scaffold 10 can contain one or more modules 30that fit in or on a rack 20 that interfaces with a tissue proximate to adefect.

A tissue scaffold module 30 can be made all or in part of abiocompatible material. For example, a module 30 can be a polymer, suchas a degradeable polymer. A module 30 can be degradable. A module 30 canbe degradable when implanted into a subject, such as a mammal. A module30 can be non-degradable. A module 30 can be formed in whole or in partof a polymer, such as a degradable polymer. Polymer materials suitablefor a module 30 can be as discussed herein.

A module 30 can have a porous microstructure in all or part of themodule 30. An internal porous microstructure of a module 30 can becreated using an image-based design technique, such as that described inU.S. Pat. No. 7,174,282.

Shape

A scaffold module 30 can have a shape designed to mimic or substantiallymimic a contour of a non-defective or healthy tissue in the region of atissue defect. A plurality of modules 30 can in combination mimic orsubstantially mimic a contour of a non-defective or healthy tissue inthe region of a tissue defect fill or substantially fill a tissuedefect. For example, a plurality of modules 30 can in combination fillor substantially fill a tissue defect such that the contours of ahealthy or non-defective tissue are provided in the region of thedefect. A scaffold module 30 can have an external shape created using animage-based design technique. Exemplary image-based design techniques sare described in U.S. Pat. No. 7,174,282, incorporated herein byreference.

A scaffold module 30 can have an irregular disk shape. A scaffold module30 having an irregular disk shape can have one or more flat orsubstantially flat sides. For example, a scaffold module 30 can have anirregular disk shape with two flat sides (see e.g., FIG. 2). A scaffoldmodule 30 can have a circumference substantially in the form of anoutline of missing or damaged tissue in, for example, a tissue gap. Aplurality of scaffold modules 30 having a circumference substantially inthe form of an outline of missing or damaged tissue can combine to fillor substantially fill a tissue defect, such as a gap (see e.g., FIG. 4).

An implantable scaffold 10 described herein can have one or more modules30. For example, a scaffold 10 can have a first module 30 a. As anotherexample, a scaffold 10 can have a second module 30 b. As anotherexample, a scaffold 10 can have a third module 30 c. As another example,a scaffold 10 can have a fourth module 30 d. As another example, ascaffold 10 can have a fifth module 30 e. As another example, a scaffold10 can have a sixth module 30. As another example, a scaffold 10 canhave a seventh module 30. As another example, a scaffold 10 can have aneighth module 30. As another example, a scaffold 10 can have a ninthmodule 30. As another example, a scaffold 10 can have a tenth module 30.As another example, a scaffold 10 can have more than ten module 30.

In a scaffold 10 with a plurality of modules 30, one or modules 30 canbe an end module 30 a or 30 e (e.g., a module proximate to tissue). Anend module 30 a or 30 e can have one or more projection that caninterface with proximate tissue. For example, an end module 30 a or 30 ecan have projection that can fit into a marrow space of a surroundingbone 40.

A scaffold 10 design can incorporate any number of modules 30 of anythickness with any necessary or desired geometric shape. Such designdoes not have to be limited to cylinders of conventional designs. It isunderstood that a scaffold 10 can have as many modules 30 as necessaryor desired to fill or substantially fill a defect. The number of modules30 of a scaffold 10 can be according to the design of the module 30(e.g., thickness) and the size of a tissue defect. The number of modules30 necessary to fill or substantially fill a tissue defect can bedetermined in advance. The number of modules 30 necessary to fill orsubstantially fill a tissue defect can be determined during a procedure,such as a surgery to correct a tissue defect.

Connectors

A scaffold module 30 can have one or more connectors 35. A connector 35of a module 30 can couple the module 30 to another component of thescaffold 10. For example, a module 30 can have a connector 35 thatcouples that module 30 to another module 30. As another example, amodule 30 can have a connector 35 that couples that module 30 to a rack20.

A module 30 can have multiple connectors 35. For example, a module 30can have multiple connectors 35 for coupling that component to one ormore other modules 30. As another example, a module 30 can have multipleconnectors 35 for coupling that module 30 to one other module 30. Asanother example, a module 30 can have multiple connectors 35 forcoupling that module 30 to a rack 20. As another example, a module 30can have multiple connectors 35 for coupling that module 30 to one ormore other modules 30 and a rack 20.

A connector 35 can permanently or removably couple a module 30 toanother component of a scaffold 10.

A connector 35 of a module 30 can have a shape suitable for connectingto another component of a scaffold 10. For example, a connector 35 canbe shaped for snap fit, mortise and tenon, dovetail or other joints. Amodule 30 can include one or more connectors 35 having the same shape ordifferent shapes. Shape of a module connector 35 can complement theshape of a connector 25 or 35 of a scaffold 10 component to be coupled.

A scaffold module 30 can include a connector(s) 35 in the form of aslot(s), e.g., a raised slot. A raised slot connector 35 of a module 30can mate with a slot in or on another module 30 or a rack 20. Methods offixing a module 30 to a rack 20 include, but are not limited to, snapfit, mortise and tenon, dovetail or similar joints. In addition tofitting within the fixation sleeve, one or more scaffold modules 30 canhave associated snap fit, mortise and tenon, dovetail or similar jointto lock together. A connector 35 can be a dovetail connector 35. Aconnector 35 can be a an elliptical dovetail connector 35.

A connector 35 can be or include a ridge that functions to connect amodule 30 to another module 30 or to a rack 20 or both. A connector 35can include a ridge designed to be melted by, for example, theapplication of energy, such as, for example, heat or ultrasound.Application of energy to the ridge can fuse the a module 30 to anothermodule 30, to a rack 20, or both.

A connector 35 can be a liquefied biocompatible polymer. A liquefiedbiocompatible polymer connector 35 can harden or fuse one module 30 toanother module 30 or to a rack 20 or both.

A first module 30 a can have an A connector 35 a. The A connector 35 aof a first module 30 a can connect to a B connector 35 b of a secondmodule 30 b. An A connector 35 a of a first module 30 a and a Bconnector 35 b of a second module 30 b can be the same type of connector35. An A connector 35 a of a first module 30 a and a B connector 35 b ofa second module 30 b can be different types of connector 35.

A scaffold module 30 can have a C connector 35 c. The C connector 35 cof a module 30 can connect to a D connector 25 of a scaffold rack 20.The C connector 35 c of a module 30, for connecting a scaffold rack 20,can be included in or on a module 30 along with other connectors 35,e.g., for connecting to other modules 30.

A scaffold module 30 can include a bioactive agent, as described furtherherein. A scaffold module 30 can include a surface modification or acoating, as described further herein. A scaffold module 30 can includeone or more cell types, as described further herein.

Scaffold Rack

Implantable scaffolds 10 described herein can include a tissue scaffoldrack 20. A scaffold 10 can comprise a rack 20 designed to accommodateone or more modules 30. The scaffold rack 20 can fit an anatomicalregion or a center core that runs between two anatomical regions. Forexample, a scaffold 10 can contain a rack 20 that fits one or moremodules 30 that interfaces with a tissue proximate to a defect. Forexample, a rack 20 can accommodate a first module 30 a and a secondmodule 30 b (or additional modules 30), which combine to fill orsubstantially fill a tissue defect.

A tissue scaffold rack 20 can be made all or in part of a biocompatiblematerial. For example, a rack 20 can be a polymer, such as a degradeablepolymer. The scaffold rack 20 can comprise a biocompatible material asdescribed herein. The scaffold rack 20 can be degradable. The scaffoldrack 20 can be degradable when implanted into a subject, such as amammal. The scaffold rack 20 can be non-degradable. The scaffold rack 20can be formed in whole or in part of a polymer, such as a degradablepolymer. Polymer materials suitable for the scaffold rack 20 can be asdiscussed herein.

A rack 20 can have a porous microstructure in all or part of the rack20. An internal porous microstructure of a rack 20 can be created usingan image-based design technique, such as that described in U.S. Pat. No.7,174,282.

An implantable scaffold 10 described herein can have a rack 20 thataccommodates one or more modules 30. For example, a rack 20 canaccommodate at least one, at least two, at least three, at least four,at least five, at least six, at least seven, at least eight, at leastnine, at least ten, or more modules 30.

Shape

A scaffold rack 20 can have a shape designed to mimic or substantiallymimic a contour of a non-defective or healthy tissue in the region of atissue defect. A rack 20 can accommodate a plurality of modules 30 thatin combination mimic or substantially mimic a contour of a non-defectiveor healthy tissue in the region of a tissue defect fill or substantiallyfill a tissue defect. For example, a rack 20 that accommodates aplurality of modules 30 can in combination fill or substantially fill atissue defect such that the contours of a healthy or non-defectivetissue are provided in the region of the defect. A scaffold rack 20 canhave an external shape created using an image-based design technique.Exemplary image-based design techniques s are described in U.S. Pat. No.7,174,282, incorporated herein by reference.

The shape of a scaffold rack 20 can be designed to span a tissue defect,such as a gap. A scaffold rack 20 can span a tissue gap in a vertebrate.The shape of a scaffold rack 20 can be designed to conform orsubstantially conform to the contours of a healthy or non-defectivetissue so as to span a tissue defect, such as a gap. For example, ascaffold rack 20 can span a bone gap. As another example, a scaffoldrack 20 can span a long bone gap. As another example, a scaffold rack 20can span a bone gap in a mammal and one or more scaffold modules 30 fillthe gap.

A scaffold rack 20 can comprise a bar, which the modules 30 at leastpartially envelop.

A scaffold rack 20 can have a trough shaped portion. A trough shapedportion of a scaffold rack 20 can have multiple (e.g., two) side regions21, a proximal end 24 and a distal end 26. A rack 20 can span a bone gap(e.g., a long bone gap) by the proximal end 24 or distal end 26 of thetrough shaped region partially or substantially enveloping the bone 40.A trough shaped portion of a scaffold rack 20 can have a bottom region22 that accommodates one or more modules 30 into the trough shapedregion by contacting the bottom region 22 and substantially spanning thetwo side regions 21.

A scaffold rack 20 can span a tissue defect, such as a gap or bone gap,in a subject, such as a vertebrate or mammalian subject. For example, ascaffold rack 20 can span a bone gap (e.g., a long bone gap) with theproximal end 24 or distal end 26 of the rack 20 partially orsubstantially enveloping the bone 40. The rack 20 thus interfaced withthe bone can accommodate one or modules 30 that fill or substantial fillthe gap. For example, a scaffold rack 20 can span a gap in the body of amandible with the proximal end 24 or distal end 26 of the rack partiallyor substantially enveloping the body of the mandible, where the rack 20accommodates one or modules 30 filling or substantially filled the gapin the mandible.

Connectors

A scaffold rack 20 can have one or more connectors 25 for connecting therack 20 to other components of the scaffold 10 or to tissue.

A scaffold rack 20 can have one or more connectors 25. A connector 25 ofa rack 20 can couple one or more modules 30 to the rack 20. One ormodules 30 can be joined to the scaffold rack 20 as described furtherherein. A connector 25 of a rack 20 can couple that rack 20 to anotherscaffold rack 20.

A connector 25 can permanently or removably couple a rack 20 to anothercomponent of a scaffold 10.

A connector 25 of a rack 20 can have a shape suitable for connecting toanother component of a scaffold 10. For example, a connector 25 can beshaped for snap fit, mortise and tenon, dovetail or other joints. A rack20 can include one or more connectors 25 having the same shape ordifferent shapes. Shape of a rack connector 25 can complement the shapeof a connector 25 or 35 of another scaffold 10 component, such as amodule 30.

A scaffold rack 20 can include a connector(s) 25 in the form of aslot(s) (see e.g., FIG. 1), e.g., a raised slot. A raised slot connector25 of a rack 20 can mate with a slots in or on one or more scaffoldmodules 30. Methods of fixing a rack 20 to a module 30 include, but arenot limited to, snap fit, mortise and tenon, dovetail or similar joints.In addition to fitting within a rack 20, one or more scaffold modules 30can have associated snap fit, mortise and tenon, dovetail or similarjoint to lock together. A rack connector 25 can be a dovetail connector25. A rack connector 25 can be a an elliptical dovetail connector 25.

A connector 25 can be or include a ridge that functions to connect arack 20 to another component of the scaffold, such as a module 30. Aconnector 25 can include a ridge designed to be melted by, for example,the application of energy. Application of energy to the ridge can fusethe rack 20 to another component of the scaffold, such as one or moremodules 30 or different rack 20, or both.

A connector 25 can be a liquefied biocompatible polymer. A liquefiedbiocompatible polymer connector 25 can harden or fuse a rack 20 toanother component of the scaffold, such as one or more modules 30.

A scaffold rack 20 can have a D connector 25. The D connector 25 of arack 20 can connect to a C connector 30 c of a module 30. The Dconnector 25 of a rack 20, for connecting a module 30, can be includedin or on a rack 20 along with other connectors 25. A plurality of Dconnectors 25 on a rack 20 can couple to one or modules 30 in or on therack 20 through, for example, a C connector 30 c of a module 30. One ormore D connectors 25 can be in the bottom 22 of a trough shaped regionof the rack 20 that couple to C connectors 30 c on the modules 30, wherethe modules 30 contact the bottom 22 of the trough shaped region.

A D connector 25 can be in a recess of the rack 20 and a C connector 30c can be in a protuberance of a module 30, wherein the protuberance fitsinto the recess. A D connector 25 can be on a protuberance of the rack20 and a C connector 30 c can be in a recess of a module 30, wherein theprotuberance fits into the recess.

A scaffold rack 20 can include a bioactive agent, as described furtherherein. A scaffold rack 20 can include a surface modification or acoating, as described further herein. A scaffold rack 20 can include oneor more cell types, as described further herein.

Scaffold Materials

A scaffold 10 described herein can include one or more componentsfabricated in whole or in part from a polymer material, such as adegradable polymer material, a porous polymer material, or a degradableporous polymer material. Suitable scaffold materials are discussed in,for example, Ma and Elisseeff, ed. (2005) Scaffolding in TissueEngineering, CRC, ISBN 1574445219; Saltzman (2004) Tissue Engineering:Engineering Principles for the Design of Replacement Organs and Tissues,Oxford ISBN 019514130X.

A scaffold 10 made in whole or in part from a polymer material can:provide structural and/or functional features of the target tissue(e.g., bone 40); allow cell attachment and migration; deliver and retaincells and biochemical factors; enable diffusion of cell nutrients andexpressed products; or exert certain mechanical and biologicalinfluences to modify the behavior of the cell phase. Scaffold materialscan be biocompatible materials that generally form a porous,microcellular matrix, which can provide a physical support or anadhesive substrate for introducing bioactive agents or cells duringfabrication, culturing, or in vivo implantation.

Generally, a biocompatible material is one which stimulates at most onlya mild, often transient, implantation response, as opposed to a severeor escalating response. A biodegradable or degradable material isgenerally understood to decomposes under normal in vivo physiologicalconditions into components which can be metabolized or excreted.

Material biodegradability can provide for absorption of the matrix bythe surrounding tissues and can eliminate the necessity of a surgicalremoval. The rate at which degradation occurs can coincide as much aspossible with the rate of tissue formation. Thus, while cells arefabricating their own natural structure around themselves (see e.g.,FIG. 6B), the scaffold 10 or components thereof can provide structuralintegrity and eventually break down leaving the neotissue, newly formedtissue which can assume the mechanical load. One or more scaffoldmaterials can be modified so as to increase biodegradability. Forexample, PCL is a biodegradable polyester by hydrolysis of its esterlinkages in physiological conditions, and can be further modified withring opening polymerization to increase its biodegradability.

Nonlimiting examples of suitable biodegradable materials includepolycaprolactone, polylactide, polyglycolide, poly(lactide-glycolide),poly(propylene fumarate), poly(caprolactone fumarate), polyethyleneglycol, and poly(glycolide-co-caprolactone), polysaccharides (e.g.alginate), chitosan, polyphosphazene, polyacrylate, polyethyleneoxide-polypropylene glycol block copolymer, fibrin, collagen,fibronectin, polyvinylpyrrolidone, hyaluronic acid, polycarbonates,polyamides, polyanhydrides, polyamino acids, polyortho esters,polyacetals, polycyanoacrylates, polyurethanes, polyacrylates,ethylene-vinyl acetate polymers and other acyl substituted celluloseacetates and derivatives thereof, and analogs, mixtures, combinationsand derivatives of any of the above

In some embodiments, a scaffold, or portion or component thereof,comprises a material having a porous microstructure. Pores of ascaffold, or portion or component thereof, can mimic internal bone 40structure, allow adherence of cells, provide an open volume for seedingof cells, provide an open volume for growth factors or other additives,allow adherence of another matrix layer, serve as conduits forvascularization, provide internal bone 40 features, or facilitateperfusion. A scaffold material with a high porosity and an adequate poresize is preferred so as to facilitate cell introduction and diffusionthroughout the whole structure of both cells and nutrients. Pores of ascaffold material can be engineered to be of various diameters. Forexample, the pores of a scaffold material can have a diameter range frommicrometers to millimeters. As another example, the pores of the matrixmaterial have a diameter of about 100 μm to about 600 μm (e.g., about150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, about400 μm, about 450 μm, about 500 μm, or about 550 μm). It is understoodthat the pores of a scaffold material can have the same, approximatelythe same, or different average diameters between different components orportions of a scaffold 10. For example, a first module 30 a can have afirst average pore diameter, a second module 30 b can have a secondaverage pore diameter, and the first average pore diameter can be thesame, approximately the same, or different than the second average porediameter. As another example, scaffold modules 30 can have a firstaverage pore diameter, a scaffold rack 20 can have a second average porediameter, and the first average pore diameter can be the same,approximately the same, or different than the second average porediameter.

A scaffold, or portion or component thereof, can be produced fromproteins (e.g. extracellular matrix proteins such as fibrin, collagen,and fibronectin), polymers (e.g., polyvinylpyrrolidone), polysaccharides(e.g. alginate), hyaluronic acid, or analogs, mixtures, combinations,and derivatives of the above.

A scaffold, or portion or component thereof, can be formed of syntheticpolymers. Such synthetic polymers include, but are not limited to,poly(ethylene) glycol, bioerodible polymers (e.g., poly(lactide),poly(glycolic acid), poly(lactide-co-glycolide), poly(caprolactone),polyester (e.g., poly-(L-lactic acid), polyanhydride, polyglactin,polyglycolic acid), polycarbonates, polyamides, polyanhydrides,polyamino acids, polyortho esters, polyacetals, polycyanoacrylates),polyphosphazene, degradable polyurethanes, non-erodible polymers (e.g.,polyacrylates, ethylene-vinyl acetate polymers and other acylsubstituted cellulose acetates and derivatives thereof), non-erodiblepolyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride,polyvinyl pyrrolidone, poly(vinylimidazole), chlorosulphonatedpolyolifins, polyethylene oxide, polyvinyl alcohol (e.g., polyvinylalcohol sponge), synthetic marine adhesive proteins, Teflon®, Nylon, orAnalogs, Mixtures, Combinations (e.g., Polyethylene Oxide-Polypropyleneglycol block copolymer; poly(D,L-lactide-co-glycolide) fiber matrix),and derivatives of the above.

A scaffold, or portion or component thereof, can be formed of naturallyoccurring polymers or natively derived polymers. Such polymers include,but are not limited to, agarose, alginate (e.g., calcium alginate gel),fibrin, fibrinogen, fibronectin, collagen (e.g., a collagen gel),gelatin, hyaluronic acid, chitin, and other suitable polymers andbiopolymers, or analogs, mixtures, combinations, and derivatives of theabove. Also, a scaffold, or portion or component thereof, can be formedfrom a mixture of naturally occurring biopolymers and syntheticpolymers.

A scaffold, or portion or component thereof, can comprise a crystallineor mineral component. For example, A scaffold, or portion or componentthereof, can include the inorganic mineral hydroxyapatite (also known ashydroxylapatite). About seventy percent of natural bone is made up ofhydroxyapatite. In some embodiments, a scaffold, or portion or componentthereof, comprises a ground natural substance containing hydroxyapatite,such as bone. In some embodiments, a scaffold, or portion or componentthereof, comprises substantially pure hydroxyapatite.

A scaffold, or portion or component thereof, can comprise a compositematerial comprising at least two components described above. As anexample, a composite scaffold material can comprise at least three, atleast four, at least five, at least six, at least seven, at least eight,at least nine, at least ten, or more, components. The plurality ofcomponents can be homogenously mixed throughout the scaffold,heterologously mixed throughout the scaffold, or separated intodifferent layers of the scaffold, or a combination thereof.

In some embodiments, a scaffold, or portion or component thereof,comprises polycaprolactone, polylactide, polyglycolide,poly(lactide-glycolide), poly(propylene fumarate), poly(caprolactonefumarate), polyethylene glycol, poly(glycolide-co-caprolactone), ormixtures thereof.

For example, a scaffold, or portion or component thereof, can be formedin whole or in part of polycaprolactone or a mixture, composite, orderivative thereof. Polycaprolactone can be a particularly usefulmaterial where the scaffolds 10 are prepared by the methods described inU.S. Pat Pub No. 2003/0069718, U.S. Pat Pub No. 2006/0276925, U.S. PatPub No. 2008/0195211, U.S. Pat Pub No. 2008/0215093, or U.S. patentapplication Ser. No. 13/036,470, all are incorporated herein byreference in their entireties.

Surface Coating

A scaffold, or portion or component thereof, described herein caninclude a surface modification or a coating. A modular scaffold 10design can allow for homogenous or heterogenous surface modificationtechniques or drug delivery.

Where a scaffold 10 is designed to fill a bone defect 45, anosteoconductive mineral coating can be utilized. An osteoconductivemineral coating can comprises a plurality of discrete mineral islands onthe scaffold, or the mineral coating can be formed on the entire surfaceof the scaffold 10. In one exemplary form, the osteoconductive mineralcoating comprises a substantially homogeneous mineral coating. In otherembodiments, the mineral coatings may be any suitable coating materialcontaining calcium and phosphate, such as hydroxyapatite,calcium-deficient carbonate-containing hydroxyapatite, tricalciumphosphate, amorphous calcium phosphate, octacalcium phosphate, dicalciumphosphate, calcium phosphate, and the like. The mineral coating may alsoinclude a plurality of layers having distinct dissolution profiles tocontrol dissolution order, kinetics and bioactive delivery properties.

To induce formation of a calcium phosphate-based mineral layer, thescaffold 10 in some embodiments is incubated in modified simulated bodyfluid (mSBF) solutions for mineral nucleation and growth. The mSBFsolution can contain ionic constituents of blood plasma, with double theconcentrations of calcium and phosphate ions, held at physiologictemperature and pH 6.8. The growth of calcium phosphate-based minerals,specifically bone-like minerals, on bioresorbable polymer matrices usingmSBF incubation has been demonstrated (Lin et al., 2004; Murphy et al.,2002, 2005).

A scaffold, or portion or component thereof, can be coated individuallyor in groups using, for example, a CaP coating technology. A scaffold,or portion or component thereof, can be modified individually or ingroups using a technique such as aminolysis for RGD attachment, chemicalconjugation, layer by layer deposition, or chemical vapor deposition.

A scaffold, or portion or component thereof, can have the same orsimilar surface modification or coating as another component. Ascaffold, or portion or component thereof, can have a different surfacemodification or coating as other components. A module 30 can have thesame or different surface modification or coating as other modules 30 ofthe scaffold 10. Thus is provided spatial control over release of growthfactors or drugs.

A scaffold, or portion or component thereof, can comprise anosteoconductive mineral coating. For example, one or more modules 30 ofa scaffold 10 can be coated with a composition comprising an scaffold,or portion or component thereof, can comprise an osteoconductive. Anosteoconductive mineral coating can include one or more of ishydroxyapatite, calcium-deficient carbonate-containing hydroxyapatite,tricalcium phosphate, amorphous calcium phosphate, octacalciumphosphate, dicalcium phosphate, calcium phosphate, or a mixture thereof.For example, an osteoconductive mineral coating can be calcium-deficientcarbonate-containing hydroxyapatite.

Cells

In various embodiments of scaffolds 10 described herein, cells can beintroduced (e.g., implanted, injected, infused, or seeded) into or ontoa scaffold, or portion or component thereof. Cells can be derived fromthe intended recipient of the scaffold, or from another donor.Additionally, the cell can be a primary cell, i.e., taken from the donorwithout culture, or the cell could be cultured any length of time priorto seeding. Further, the cage can be seeded with cells then incubatedunder appropriate conditions to allow colonization of the cage to anydegree prior to implant.

Different types of cells can be co-introduced or sequentiallyintroduced. Where differing types of cells are employed, they can beintroduced in the same spatial position, similar spatial positions, ordifferent spatial positions, relative to each other.

Cells can be introduced into the scaffold, or portion or componentthereof, by a variety of means known to the art. Methods for theintroduction (e.g., infusion, seeding, injection, etc.) of cells into orinto the scaffold material are discussed in, for example, Ma andElisseeff, ed. (2005) Scaffolding In Tissue Engineering, CRC, ISBN1574445219; Saltzman (2004) Tissue Engineering: Engineering Principlesfor the Design of Replacement Organs and Tissues, Oxford ISBN019514130X; Minuth et al. (2005) Tissue Engineering: From Cell Biologyto Artificial Organs, John Wiley & Sons, ISBN 3527311866. For example,cells can be introduced into or onto the matrix by methods includinghydrating freeze-dried scaffolds 10 with a cell suspension (e.g., at aconcentration of 100 cells/ml to several million cells/ml). Methods ofaddition of additional agents vary, as discussed below.

Cells can be introduced into or onto a scaffold material at the time offabrication. For example, cells can be introduced into the scaffold 10by a bioplotter, or other similar device, during or near the time whenbiocompatible polymer layers are formed into a 3-dimensional scaffold 10(e.g., cell printing).

Cells can be introduced into or onto individual modules 30 prior toassembly or joining of the modules 30 to one another or a scaffold rack20.

Methods of culturing and differentiating cells in or on scaffolds 10 aregenerally known in the art (see e.g., Saltzman (2004) TissueEngineering: Engineering Principles for the Design of Replacement Organsand Tissues, Oxford ISBN 019514130X; Vunjak-Novakovic and Freshney, eds.(2006) Culture of Cells for Tissue Engineering, Wiley-Liss, ISBN0471629359; Minuth et al. (2005) Tissue Engineering: From Cell Biologyto Artificial Organs, John Wiley & Sons, ISBN 3527311866). As will beappreciated by one skilled in the art, the time between cellintroduction into or onto the scaffold 10 and engrafting the resultingscaffold 10 can vary according to particular application. Incubation(and subsequent replication and/or differentiation) of the engineeredcomposition cells in or on the scaffold material can be, for example, atleast in part in vitro, substantially in vitro, at least in part invivo, or substantially in vivo. Determination of optimal culture time iswithin the skill of the art. A suitable medium can be used for in vitroprogenitor cell infusion, differentiation, or cell transdifferentiation(see e.g., Vunjak-Novakovic and Freshney, eds. (2006) Culture of Cellsfor Tissue Engineering, Wiley-Liss, ISBN 0471629359; Minuth et al.(2005) Tissue Engineering: From Cell Biology to Artificial Organs, JohnWiley & Sons, ISBN 3527311866). The culture time can vary from about anhour, several hours, a day, several days, a week, or several weeks. Thequantity and type of cells present in the matrix can be characterizedby, for example, morphology by ELISA, by protein assays, by geneticassays, by mechanical analysis, by RT-PCR, and/or by immunostaining toscreen for cell-type-specific markers (see e.g., Minuth et al. (2005)Tissue Engineering: From Cell Biology to Artificial Organs, John Wiley &Sons, ISBN 3527311866).

For small scaffolds 10 (<100 cubic millimeters in size), in vitro mediumcan be changed manually, and additional agents added periodically (e.g.,every 3-4 days). For larger scaffolds 10, the culture can be maintained,for example, in a bioreactor system, which may use a minipump for mediumchange. The minipump can be housed in an incubator, with fresh mediumpumped to the matrix material of the scaffold 10. The medium circulatedback to, and through, the matrix can have about 1% to about 100% freshmedium. The pump rate can be adjusted for optimal distribution of mediumand/or additional agents included in the medium. The medium deliverysystem can be tailored to the type of tissue or organ beingmanufactured. All culturing can be performed under sterile conditions.

The present teachings include methods for optimizing the density ofcells so as to maximize the regenerative outcome of an implantedscaffold 10. Cell densities in a scaffold 10 can be monitored over timeand at end-points. Tissue properties can be determined, for example,using standard techniques known to skilled artisans, such as histology,structural analysis, immunohistochemistry, biochemical analysis, andmechanical properties. As will be recognized by one skilled in the art,the cell densities of cells can vary according to, for example, celltype, tissue or organ type, scaffold material, scaffold volume, infusionmethod, seeding pattern, culture medium, growth factors, incubationtime, incubation conditions, and the like. Generally, cell density in ascaffold, or portion or component thereof, can be, independently, from0.0001 million cells (M) ml⁻¹ to about 1000 M ml⁻¹. For example, cellscan be present in the scaffold, or portion or component thereof, at adensity of about 0.001 M ml⁻¹, 0.01 M ml⁻¹, 0.1 M ml⁻¹, 1 M ml⁻¹, 5 Mml⁻¹, 10 M ml⁻¹, 15 M ml⁻¹, 20 M ml⁻¹, 25 M ml⁻¹, 30 M ml⁻¹, 35 M ml⁻¹,40 M ml⁻¹, 45 M ml⁻¹, 50 M ml⁻¹, 55 M ml⁻¹, 60 M ml⁻¹, 65 M ml⁻¹, 70 Mml⁻¹, 75 M ml⁻¹, 80 M ml⁻¹, 85 M ml⁻¹, 90 M ml⁻¹, 95 M ml⁻¹, 100 M ml⁻¹,200 M ml⁻¹, 300 M ml⁻¹, 400 M ml⁻¹, 500 M ml⁻¹, 600 M ml⁻¹, 700 M ml⁻¹,800 M ml⁻¹, or 900 M ml⁻¹. It is contemplated that cells can be presentin the scaffold, or portion or component thereof, in a range from onedensity value recited above to another density value recited above.

A cell included in or on a scaffold, or portion or component thereof,can be a vertebrate cell. A cell included in or on a scaffold, orportion or component thereof, can be a mammalian cell. A cell includedin or on a scaffold, or portion or component thereof, can be a stemcell. A cell included in or on a scaffold, or portion or componentthereof, can be an embryonic stem cell. A cell included in or on ascaffold, or portion or component thereof, can be an adult stem cell. Acell included in or on a scaffold, or portion or component thereof, canbe a mesenchymal stem cell. A cell included in or on a scaffold, orportion or component thereof, can be an induced pluripotent stem cell.

In some embodiments, the cell is a terminally differentiated cell, e.g.,an osteoblast, a chondrocyte, an adipose cell, a pancreatic beta cell, amuscle cell (skeletal, smooth or cardiac), a hepatocyte, or a kidneycell. In other embodiments, the cell is less differentiated, for examplea stem cell such as an embryonic stem cell or an adult stem cell, e.g.,a mesenchymal stem cell, a hematopoetic stem cell, or an endothelialstem cell. In various embodiments, the stem cell is derived from a cellisolated in the undifferentiated state. In alternative embodiments, thestem cell is induced (known as induced pluripotent stem cells or iPScells) from a differentiated cell, by any means known in the art (e.g.,by transfection with a transgene or by treatment with a cytokine).

Bioactive Agents

In some embodiments, methods and compositions described herein canfurther comprise additional agents introduced into or onto the scaffold,or portion or component thereof.

In some embodiments, the scaffold 10 comprises a bioactive agent. Abioactive agent as used herein includes, without limitation,physiologically or pharmacologically active substances that act locallyor systemically in the body. A bioactive agent can be a substance usedfor the treatment, prevention, diagnosis, cure or mitigation of diseaseor illness, or a substance which affects the structure or function ofthe body or which becomes biologically active or more active after ithas been placed in a predetermined physiological environment. Bioactiveagents include, without limitation, enzymes, organic catalysts, nucleicacids including ribozymes and antisense RNA or DNA, organometallics,proteins, demineralized bone matrix, bone marrow aspirate,glycoproteins, peptides, polyamino acids, antibodies, nucleic acids,steroidal molecules, antibiotics, antimycotics, cytokines, fibrin,collagen, fibronectin, vitronectin, hyaluronic acid, growth factors,carbohydrates, statins, oleophobics, lipids, extracellular matrix and/orits individual components, pharmaceuticals, and therapeutics.

Various agents that can be introduced include, but are not limited to,bioactive molecules, biologic drugs, diagnostic agents, andstrengthening agents.

A scaffold, or portion or component thereof, can have the same orsimilar bioactive agent(s) as another component. A scaffold, or portionor component thereof, can have a different bioactive agent(s) as othercomponents. A module 30 can have the same or different bioactiveagent(s) as other modules 30 of the scaffold 10.

The scaffold, or portion or component thereof, can comprise at least onebioactive agent. In some embodiments, cells of the scaffold 10 can be,for example, genetically engineered to express the bioactive agent orthe bioactive agent can be added to the scaffold 10. The scaffold, orportion or component thereof, can also be cultured in the presence ofthe bioactive agent. A bioactive agent can be added prior to, during, orafter cells (when present) are introduced to the scaffold, or portion orcomponent thereof. A bioactive agent can be present in an amount thatinduces ossification.

The scaffold, or portion or component thereof, can include a bioactiveagent that induces ossification. For example, a scaffold, or portion orcomponent thereof, can include a growth factor (e.g., a growth factorthat can induce ossification). As another example, a scaffold, orportion or component thereof, can include an osteoinductive cytokine.

A bioactive agent of the scaffold, or portion or component thereof, canbe bone morphogenetic protein (BMP), demineralized bone matrix, bonemarrow aspirate, transforming growth factor, fibroblast growth factor,an insulin-like growth factor, platelet derived growth factor, vascularendothelial growth factor, growth and development factor-5, plateletrich plasma, or a mixture thereof.

For example, bioactive agent of the scaffold, or portion or componentthereof, can be BMP2 or BMP7

Non-limiting examples of bioactive molecules include activin A,adrenomedullin, aFGF, ALK1, ALK5, ANF, angiogenin, angiopoietin-1,angiopoietin-2, angiopoietin-3, angiopoietin-4, angiostatin,angiotropin, angiotensin-2, AtT20-ECGF, betacellulin, bFGF, B61, bFGFinducing activity, cadherins, CAM-RF, cGMP analogs, ChDI, CLAF,claudins, collagen, collagen receptors α₁β₁ and α₂β₁, connexins, Cox-2,ECDGF (endothelial cell-derived growth factor), ECG, ECI, EDM, EGF,EMAP, endoglin, endothelins, endostatin, endothelial cell growthinhibitor, endothelial cell-viability maintaining factor, endothelialdifferentiation shpingolipid G-protein coupled receptor-1 (EDG1),ephrins, Epo, HGF, TNF-alpha, TGF-beta, PD-ECGF, PDGF, IGF, IL8, growthhormone, fibrin fragment E, FGF-5, fibronectin, fibronectin receptorα₅β₁, Factor X, HB-EGF, HBNF, HGF, HUAF, heart derived inhibitor ofvascular cell proliferation, IFN-gamma, IL1, IGF-2 IFN-gamma, integrinreceptors (e.g., various combinations of α subunits (e.g., α₁, α₂, α₃,α₄, α₅, α₆, α₇, α₈, α₉, α_(E), α_(V), α_(IIb), α_(L), α_(M), α_(X)) andβ subunits (e.g., β₁, β₂, β₃, β₄, β₅, β₆, β₇, and β₈)), K-FGF, LIF,leiomyoma-derived growth factor, MCP-1, macrophage-derived growthfactor, monocyte-derived growth factor, MD-ECI, MECIF, MMP 2, MMP3,MMP9, urokiase plasminogen activator, neuropilin (NRP1, NRP2),neurothelin, nitric oxide donors, nitric oxide synthases (NOSs), notch,occludins, zona occludins, oncostatin M, PDGF, PDGF-B, PDGF receptors,PDGFR-β, PD-ECGF, PAI-2, PD-ECGF, PF4, P1GF, PKR1, PKR2, PPAR-gamma,PPARγ ligands, phosphodiesterase, prolactin, prostacyclin, protein S,smooth muscle cell-derived growth factor, smooth muscle cell-derivedmigration factor, sphingosine-1-phosphate-1 (S1P1), Syk, SLP76,tachykinins, TGF-β, Tie 1, Tie2, TGF-β receptors, TIMPs, TNF-alpha,TNF-beta, transferrin, thrombospondin, urokinase, VEGF-A, VEGF-B,VEGF-C, VEGF-D, VEGF-E, VEGF, VEGF₁₆₄, VEGI, EG-VEGF, VEGF receptors,PF4, 16 kDa fragment of prolactin, prostaglandins E1 and E2, steroids,heparin, 1-butyryl glycerol (monobutyrin), and nicotinic amide. In otherpreferred embodiments, the matrix includes a chemotherapeutic agent orimmunomodulatory molecule. Such agents and molecules are known to theskilled artisan.

In some embodiments, the bioactive agent is a growth factor such asgrowth hormone (GH); parathyroid hormone (PTH, including PTH1-34); bonemorphogenetic proteins (BMPs), such as BMP2A, BMP2B, BMP3, BMP4, BMP5,BMP6, BMP7 and BMP8; transforming growth factor-α (TGF-α), TGF-β1 andTGF-β2; fibroblast growth factor (FGF), granulocyte/macrophage colonystimulating factor (GMCSF), epidermal growth factor (EGF), plateletderived growth factor (PDGF), growth and development factor-5 (GDF-5),an insulin-like growth factor (IGF), leukemia inhibitory factor (LIF),vascular endothelial growth factor (VEGF), basic fibroblast growthfactor (bFGF), platelet derived growth factor (PDGF), angiogenin,angiopoietin-1, del-1, follistatin, granulocyte colony-stimulatingfactor (G-CSF), hepatocyte growth factor/scatter factor (HGF/SF),interleukin-8 (IL-8), leptin, midkine, placental growth factor,platelet-derived endothelial cell growth factor (PD-ECGF),platelet-derived growth factor-BB (PDGF-BB), pleiotrophin (PTN),progranulin, proliferin, tumor necrosis factor-α (TNF-α), vascularendothelial growth factor (VEGF), a matrix metalloproteinase (MMP),angiopoietin 1 (ang1), ang2, or delta-like ligand 4 (DLL4).

In some embodiments, particularly where the scaffold 10 is to fill abone defect 45, the bioactive agent is a BMP such as BMP2, BMP4, BMP7,or BMP14, an IGF, an FGF, a PDGF, GDF-5, a TGF, a VEGF or platelet richplasma (PRP).

Drugs that can be added to the compositions of the application includeimmunomodulators and other biological response modifiers. A biologicalresponse modifier can encompass a biomolecule (e.g., peptide, peptidefragment, polysaccharide, lipid, antibody) that is involved in modifyinga biological response, such as the immune response or tissue growth andrepair, in a manner which enhances a particular desired therapeuticeffect, for example, the cytolysis of bacterial cells or the growth oftissue-specific cells or vascularization. Drugs can also be incorporateddirectly into the matrix component. Those of skill in the art will know,or can readily ascertain, other substances which can act as suitablenon-biologic and biologic drugs.

Bioactive molecules and biomolecules can be incorporated into thescaffold, or portion or component thereof, causing such to be imbeddedwithin. Alternatively, chemical modification methods may be used tocovalently link a molecule or biomolecule on the surface of thescaffold, or portion or component thereof. The surface functional groupsof the scaffold, or portion or component thereof can be coupled withreactive functional groups of the molecules or biomolecules to formcovalent bonds using coupling agents well known in the art such asaldehyde compounds, carbodiimides, and the like. Additionally, a spacermolecule can be used to gap the surface reactive groups and the reactivegroups of the molecules or biomolecules to allow more flexibility ofsuch molecules on the surface of the scaffold, or portion or componentthereof. Other similar methods of attaching molecules or biomolecules tothe interior or exterior of a scaffold, or portion or component thereof,will be known to one of skill in the art.

A scaffold 10 described herein can also be modified to incorporate adiagnostic agent, such as a radiopaque agent. The presence of suchagents can allow a physician to monitor the progression of healing prgrowth occurring internally. Such compounds include barium sulfate aswell as various organic compounds containing iodine. Examples of theselatter compounds include iocetamic acid, iodipamide, iodoxamatemeglumine, iopanoic acid, as well as diatrizoate derivatives, such asdiatrizoate sodium. Other contrast agents which can be utilized in thecompositions of the application can be readily ascertained by those ofskill in the art and may include the use of radiolabeled fatty acids oranalogs thereof.

Concentration of an agent in a scaffold, or portion or componentthereof, will vary with the nature of the compound, its physiologicalrole, and desired therapeutic or diagnostic effect. A therapeuticallyeffective amount is generally a sufficient concentration of therapeuticagent to display the desired effect without undue toxicity. Adiagnostically effective amount is generally a concentration ofdiagnostic agent which is effective in allowing the monitoring of theintegration of the scaffold, while minimizing potential toxicity. In anyevent, the desired concentration in a particular instance for aparticular compound is readily ascertainable by one of skill in the art.

A scaffold, or portion or component thereof, can be enhanced, orstrengthened, through the use of such supplements as human serum albumin(HSA), hydroxyethyl starch, dextran, or combinations thereof. Thesolubility of the scaffold materials or compositions therein can beenhanced by the addition of a nondenaturing nonionic detergent, such aspolysorbate 80. Suitable concentrations of these compounds for use inthe compositions of the application will be known to those of skill inthe art, or can be readily ascertained without undue experimentation.Scaffold materials or compositions therein can be enhanced by the use ofoptional stabilizers or diluent. The proper use of these would be knownto one of skill in the art, or can be readily ascertained without undueexperimentation.

In some embodiments, the bioactive agent is released quickly from thescaffold 10 after implantation. In other embodiments, the bioactiveagent can be formulated or bound to the scaffold 10 to be releasedslowly. For example, mineral coated microspheres can affect the releaserate of a bioactive agent from a calcium phosphate coating, includingbuilding up layers of the coating with different dissolution patterns,or binding a component to the coating that provides a functional groupto which the bioactive agent can be covalently bound. The agent can alsobe covalently or noncovalently bound to any region of the scaffoldmaterial itself to effect slow release, by any means known in the art.

Bioactive agents can be introduced into or onto the scaffold, or portionor component thereof, via a carrier based system, such as anencapsulation vehicle. For example, growth factors can bemicro-encapsulated to provide for enhanced stability or prolongeddelivery. Encapsulation vehicles include, but are not limited to,microparticles, liposomes, microspheres, or the like, or a combinationof any of the above to provide the desired release profile in varyingproportions. Other methods of controlled-release delivery of agents willbe known to the skilled artisan. Moreover, these and other systems canbe combined or modified to optimize the integration/release of agentswithin the scaffold 10.

Carrier based systems for incorporation of various agents into or ontothe scaffold 10 can: provide for enhanced intracellular delivery; tailorbiomolecule/agent release rates; increase or accelerate functionalintegration of layers; increase the proportion of agent that reaches itssite of action; improve the transport of the agent to its site ofaction; allow co-localized deposition with other agents or excipients;improve the stability of the agent in vivo; prolong the residence timeof the agent at its site of action by reducing clearance; decrease thenonspecific delivery of the agent to non-target tissues; decreaseirritation caused by the agent; decrease toxicity due to high initialdoses of the agent; alter the immunogenicity of the agent; decreasedosage frequency; or improve shelf life of the product.

Polymeric microspheres can be produced using naturally occurring orsynthetic polymers and are particulate systems in the size range of 0.1to 500 μm. Polymeric micelles and polymeromes are polymeric deliveryvehicles with similar characteristics to microspheres and can alsofacilitate encapsulation and matrix integration of the agents describedherein. Fabrication, encapsulation, and stabilization of microspheresfor a variety of payloads are within the skill of the art (see e.g.,Varde & Pack (2004) Expert Opin. Biol. 4(1) 35-51). Release rate ofmicrospheres can be tailored by type of polymer, polymer molecularweight, copolymer composition, excipients added to the microsphereformulation, and microsphere size. Polymer materials useful for formingmicrospheres include PLA, PLGA, PLGA coated with DPPC, DPPC, DSPC, EVAc,gelatin, albumin, chitosan, dextran, DL-PLG, SDLMs, PEG (e.g., ProMaxx),sodium hyaluronate, diketopiperazine derivatives (e.g., Technosphere),calcium phosphate-PEG particles, and/or oligosaccharide derivative DPPG(e.g., Solidose). Encapsulation can be accomplished, for example, usinga water/oil single emulsion method, a water-oil-water double emulsionmethod, or lyophilization. Several commercial encapsulation technologiesare available (e.g., ProLease®, Alkerme).

Polymeric hydrogels can be used to integrate various agents into thescaffold 10. For example, a polymeric hydrogel including one or moreagents can be introduced into pores of the scaffold 10.

“Smart” polymeric carriers can be used to integrate agents with thescaffold 10 (see generally, Stayton et al. (2005) Orthod CraniofacialRes 8, 219-225; Wu et al. (2005) Nature Biotech (2005) 23(9),1137-1146). Carriers of this type utilize polymers that are hydrophilicand stealth-like at physiological pH, but become hydrophobic andmembrane-destabilizing after uptake into the endosomal compartment(i.e., acidic stimuli from endosomal pH gradient) where they enhance therelease of the cargo molecule into the cytoplasm. Design of the smartpolymeric carrier can incorporate pH-sensing functionalities,hydrophobic membrane-destabilizing groups, versatile conjugation and/orcomplexation elements to allow the drug incorporation, and an optionalcell targeting component. Polymeric carriers include, for example, thefamily of poly(alkylacrylic acid) polymers, specific examples includingpoly(methylacrylic acid), poly(ethylacrylic acid) (PEAA),poly(propylacrylic acid) (PPAA), and poly(butylacrylic acid) (PBAA),where the alkyl group is progressively increased by one methylene group.Various linker chemistries are available to provide degradableconjugation sites for proteins, nucleic acids, and/or targetingmoieties. For example, pyridyl disulfide acrylate (PDSA) monomer allowefficient conjugation reactions through disulfide linkages that can bereduced in the cytoplasm after endosomal translocation of the agent(s).

Liposomes can be used to integrate agents with the scaffold 10. Theagent carrying capacity and release rate of liposomes can depend on thelipid composition, size, charge, drug/lipid ratio, and method ofdelivery. Conventional liposomes are composed of neutral or anioniclipids (natural or synthetic). Commonly used lipids are lecithins suchas (phosphatidylcholines), phosphatidylethanolamines (PE),sphingomyelins, phosphatidylserines, phosphatidylglycerols (PG), andphosphatidylinositols (PI). Liposome encapsulation methods are commonlyknown in the arts (Galovic et al. (2002) Eur. J. Pharm. Sci. 15,441-448; Wagner et al. (2002) J. Liposome Res. 12, 259-270). Targetedliposomes and reactive liposomes can also be used in combination withthe agents and matrix. Targeted liposomes have targeting ligands, suchas monoclonal antibodies or lectins, attached to their surface, allowinginteraction with specific receptors and/or cell types. Reactive orpolymorphic liposomes include a wide range of liposomes, the commonproperty of which is their tendency to change their phase and structureupon a particular interaction (eg, pH-sensitive liposomes) (see e.g.,Lasic (1997) Liposomes in Gene Delivery, CRC Press, Fla.).

Toxicity and therapeutic efficacy of agents discussed herein can bedetermined by standard pharmaceutical procedures in cell cultures and/orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where large therapeutic indices are preferred

Manufacture

In various aspects of the application, biocompatible scaffold materialsare fabricated into modules 30 or racks 20 as described above.Fabrication of biocompatible scaffold materials into shaped3-dimensional scaffold components, such as a module 30 or a rack 20, canbe according to a variety of methods known to the art. Scaffolds 10 canbe prepared according to methods described in U.S. Pat Pub No.2003/0069718, U.S. Pat Pub No. 2006/0276925, U.S. Pat Pub No.2008/0195211, or U.S. Pat Pub No. 2008/0215093, each incorporated hereinby reference.

Scaffold synthesis techniques include, but are not limited to, nanofiberself-assembly (e.g., hydrogel scaffolds), textile technologies (e.g.,non-woven polyglycolide structures), solvent casting and particulateleaching, gas foaming, emulsification/freeze-drying, thermally inducedphase separation, CAD/CAM technologies, or a combination of thesetechniques. For example, biocompatible scaffold materials can befabricated into a shaped 3-dimensional module 30 or rack 20 via computeraided design/manufacturing (CAD/CAM) technologies.

A scaffold, or portion or component thereof, described herein can bemanufactured using a range of solid free-form fabrication techniquesincluding, but not limited to, laser sintering. Another method ofmanufacturing a scaffold, or portion or component thereof, describedherein is a molding process. A scaffold, a module 30, a rack 20, or asleeve can be manufactured individually. An exemplary modular mandiblescaffold 10 manufactured from a degradable polymer polycaprolactone isdepicted in FIG. 4.

In CAD/CAM technologies of scaffold fabrication, first athree-dimensional structure is designed using computer aided design(CAD) software and then the scaffold 10 is generated by computer aidedmanufacture (CAM) process. CAM processes for scaffold fabricationinclude, for example, using ink-jet printing of polymer powders (e.g.,Bioplotter, Envisiontec, Gladbeck, Germany) or through rapid prototypingtechnology such as fused deposition modeling (FDM). Scaffold fabricationusing a bioplotter, or similar device, provides the advantage ofco-deposition of live cells (e.g., stem cells and other cells describedherein). For example, multiple printing/deposition heads can be used inthe fabrication of materials, co-deposition of cells, and/or addition ofagents such as growth factors and the like so as to provide for afabricated scaffold 10 with internal porosity features and seeded cellsor additional agents within the scaffold material or its pores.

Scaffold fabrication via CAD/CAM technologies can employ 3-dimensionaldata of the target hard tissue. As described above, the image data canbe obtained from a subject's own tissue or from similar tissue fromother than the subject. Software can import 3-dimensional volume dataand generate a plotting pathway for deposition of the scaffold material.For example, dxf-data can be prepared by processing CT scanned images orobtained from medical CAD programs like VOXIM or MIMICS, whichreconstructs a 3D model from DICOM images. The 3-dimensional model canbe an integral solid of which body surrounded by surface objects. Once a3-dimensional volume data file (e.g., a dxf file) is constructed, thesize, alignment, and position is adjusted per the dispensing layouts andchannel configurations. Such adjustment is within the ordinary skill inthe art. In a typical procedure, a selected scaffold polymer material isplaced inside the container of the dispensing module 30, and the module30 heated to a pre-optimized temperature to keep the polymer melted withappropriate viscosity for dispensing. The polymer solution can also beprepared using a solvent. With solvent, the desired viscosity can becontrolled by concentration of solute, and in some embodiments, no heatis required. The polymer solution can be dispensed in air or in liquid,optionally with chemicals required for solidification. For example,melted PCL can be dispensed in air.

For CAM fabrication techniques, the pore size of the resulting scaffold10 can be determined by distance between strands. The strand size can bedetermined by, for example, viscosity of solution, needle innerdiameter, and dispensing speed. Preferably, pore size parameters aredetermined prior to fabrication of a 3-dimensional structure, as iswithin the skill of the art.

Use

Various embodiments of the scaffolds 10 described herein holdsignificant clinical value because of their modular design,biomaterials, anatomic shape, and interior structural features. Variousscaffolds 10 of the present disclosure can provide multiple modularinserts of all degradable materials along with optional incorporation ofcells, osteoinductive coatings, or release of bioactive agents. It isthese features, at least in part, which sets the tissue modules 30disclosed herein apart from other conventional tissue defect treatmentoptions.

Another provided aspect is a method of treating a tissue defect in asubject by implanting a tissue scaffold 10 described herein into asubject in need thereof. A determination of the need for treatment willtypically be assessed by a history and physical exam consistent with thetissue defect at issue. Subjects with an identified need of therapyinclude those with a diagnosed tissue defect. The subject is preferablyan animal, including, but not limited to, mammals, reptiles, and avians,more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, andmost preferably human.

As an example, a subject in need may have damage to a tissue, and themethod provides an increase in biological function of the tissue by atleast 5%, 10%, 25%, 50%, 75%, 90%, 100%, or 200%, or even by as much as300%, 400%, or 500%. As yet another example, the subject in need mayhave a disease, disorder, or condition, and the method provides anengineered tissue scaffold 10 sufficient to ameliorate or stabilize thedisease, disorder, or condition. For example, the subject may have adisease, disorder, or condition that results in the loss, atrophy,dysfunction, or death of cells. Exemplary treated conditions includearthritis; osteoarthritis; osteoporosis; osteochondrosis;osteochondritis; osteogenesis imperfecta; osteomyelitis; osteophytes(i.e., bone spurs); achondroplasia; costochondritis; chondroma;chondrosarcoma; herniated disk; Klippel-Feil syndrome; osteitisdeformans; osteitis fibrosa cystica, a congenital defect that results inthe absence of a tissue; accidental tissue defect or damage such asfracture, wound, or joint trauma; an autoimmune disorder; diabetes(e.g., Charcot foot); cancer; a disease, disorder, or condition thatrequires the removal of a tissue (e.g., tumor resection); and/or adisease, disorder, or condition that affects the trabecular to corticalbone ratio. For example, a modular tissue scaffold 10 described hereincan be implanted in a subject who would otherwise need to undergo anosteochondral autograft. In a further example, the subject in need mayhave an increased risk of developing a disease, disorder, or conditionthat is delayed or prevented by the method.

Implantation of a tissue scaffold 10 described herein is within theskill of the art. For example, a scaffold rack 20 can be fixed to atissue site flanking or surrounding a defect using a range of surgicalfixation techniques including, but not limited to, degradable fixationpins sonically welded through the fixation sleeve or post into thesurrounding tissue to form a bond; a metal screw fixation where screwsare used to fix the mesh; or a degradable screw fixation where screwsare used to fix the mesh.

A scaffold 10 described can be coupled with or attached to tissue,directly or indirectly, including, but not limited to, using screws,welding, press or snap fit, or fasteners, such as, for example, made ofmetal, plastic or some other material. Means such as a central pin,screw, or rod in a tunnel can be used to connect a scaffold, orcomponents thereof, into a tissue, such as bone 40. As another example,a key that can be expanded can join the scaffold 10 and a tissue, suchas bone 40. As other examples, a scaffold 10 and a tissue can be joinedin situ according to welding, UV, glue, or thermosetting. A scaffold 10described herein can be anchored to other tissues, such as ligaments.Welding can be accomplished by the application of energy, such as, forexample, heat, ultrasonic or some other method.

The scaffold 10 assembly can be either fully or partially implanted intoa tissue of the subject to become a functioning part thereof. Thescaffold 10 can initially attaches to and communicates with the hostthrough a cellular monolayer. In some embodiments, over time, theintroduced cells (where present) can expand and migrate out of thepolymeric scaffold material matrix to the surrounding tissue. Afterimplantation, cells surrounding the tissue scaffold 10 can enter throughcell migration. The cells surrounding the tissue module 30 can beattracted by biologically active materials, including biologicalresponse modifiers, such as polysaccharides, proteins, peptides, genes,antigens, and antibodies which can be selectively incorporated into thematrix to provide the needed selectivity, for example, to tether thecell receptors to the scaffold 10 or stimulate cell migration into thescaffold, or both. Generally, the scaffold material is porous, allowingfor cell migration, augmented by both biological and physical-chemicalgradients. One of skill in the art will recognize and know how to useother biologically active materials that are appropriate for attractingcells to the matrix.

The methods, compositions, and devices of the application can includeconcurrent or sequential treatment with one or more of enzymes, ions,growth factors, and biologic agents, such as thrombin and calcium, orcombinations thereof. The methods, compositions, and devices of theapplication can include concurrent or sequential treatment withnon-biologic and/or biologic drugs.

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

All publications, patents, patent applications, and other referencescited in this application are incorporated herein by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application or other reference wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes. Citation of a reference herein shallnot be construed as an admission that such is prior art to the presentdisclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1

This example describes implantation of a bioresorbable PCL scaffold,resorbable CaP coating, and coated resorbable PCL fixation pins.

A 3.5 cm mandibular segmental defect was surgically created in a anadult Yorkshire pig model (see FIG. 5A). Initial sizing of scaffoldsleeve with pins (blue circle) located in drill holes is shown in FIG.5B (rectangular highlights on left and right of image). A completewelded scaffold bioresorbable pins were implanted in the surgical defectof the adult Yorkshire pig model. Final scaffold implantation andwelding of modules (rectangular highlight in upper center of image) andwelding of pins (rectangular highlights on left and right of image) areshown in FIG. 5C. The modules shown in FIG. 5C may be mechanicallyintegrated by ultrasonic means, thermal welding, or gluing. Mechanicallyintegrating the modules increases the interface strength between thefirst module and the second module.

Results showed that 7 months post-surgery, the pigs receiving theimplants retained complete masticatory function, being able to eat anormal diet. CT scans of pigs at 6 months post-surgery show that thescaffolds support masticatory loads and allow bone 40 formation withbridging of the defect and some bone 40 fill in the scaffold (see e.g.,FIG. 6). Three dimensional reconstruction in FIG. 6A shows bone 40bridging the original defect (defect margins shown by vertical lines).As seen in FIG. 6A, the mandible after 6 months is in a correct anatomicposition. Pore structure and bone 40 growth in the scaffold module isshown in FIG. 6B (rectangular highlight). Bone 40 fill in a second pigimplanted with the scaffold is shown in FIG. 6C (rectangular highlight).

This demonstrates that the completely resorbable scaffold withresorbable fixation can withstand in vivo mastication loads withoutfailure.

Bioresorbable pin fixation in vivo after 6 months post-surgery are shownin FIG. 7. Dual pin tracts in mandible are shown in FIG. 7A (tractsoutlined with solid line). FIG. 7B is a slice image 1.2 mm from the viewin FIG. 7A, showing bone 40 formation (circular highlight) underneaththe lower pin tract.

The above data validates an embodiment of a platform resorbablemandibular reconstruction system. First, the modular platform systemwelded in the OR has sufficient strength to withstand masticatory loads,and it maintained fixation to the mandible, allowing the pigs tomasticate normally. Second, the scaffolds support bone 40 formation evenin the absence of a biologic. It is presently expected that addition ofa an osteobiologic can result in even more rapid and complete bone 40regeneration.

Example 2

This example describes design and implantation of a modular scaffold forplacement of amodular mandibular reconstruction scaffold within amandibular defect in a pig mandible.

FIG. 8 shows a medial-lateral view of a modular scaffold design 800 withmarrow space interfaces that mechanically stabilize the endoprosthesisin addition to pins that are placed through the marrow space interfaces.Three modules 802, 804, and 806 are shown in FIG. 8 with pins 808, 810,and 812 providing additional fixation. The modules 802, 804, and 806interface with cut bone ends protruding into the marrow space. The pins808, 810, and 812 provide additional fixation for the scaffold modules.

FIG. 9 shows an anterior-posterior view of modular scaffold design 800from FIG. 8 with associated pins. The anterior-posterior view of modularscaffold design 800 shows scaffolds 802 and 804, and associated pins 808and 812.

FIG. 10 shows placement of a modular mandibular reconstruction scaffold1000 corresponding to modular scaffold design 800 within a mandibulardefect 1002 in a pig mandible 1004. This illustration shows how themodular scaffold stabilizes the mandibular defect and interfaces withthe marrow space of the mandible.

REFERENCES

-   Hollister, S. J. Scaffold engineering: a bridge to where?    Biofabrication 1:012001 (2009).-   Lin et al. “A novel method for internal architecture design to match    bone elastic properties with desired porosity”, Journal of    Biomechanics 37:623-36, 2004.-   Murphy et al., “Bioinspired growth of crystalline carbonate apatite    on biodegradable polymer substrata”, J Am Chem Soc 124:1910-7, 2002.-   Murphy et al., “Effects of a bone-like mineral film on phenotype of    adult human mesenchymal stem cells in vitro”, Biomaterials    26:303-10, 2005.

The invention claimed is:
 1. A biocompatible system for filling a tissuegap comprising: a first module that is biocompatible and degradable anda second module that is biocompatible and degradable; wherein one ofeach module has an irregular disk shape having two flat sides and eachmodule comprises a dovetail connector on each flat side, wherein thedovetail connectors from one of the first or second module is designedto couple with one of the dovetail connectors from the other of thefirst or second module and wherein the circumference of the irregulardisk correspond to a shape of the gap being filled by the tissuescaffold; a biocompatible and degradable tissue scaffold rack comprisinga trough shaped portion having two side regions, a bottom region, aproximal end and a distal end, wherein the tissue scaffold rackcomprises a first coupling portion having a first shape and the firstmodule comprises a second coupling portion having a second shape thatcorresponds to the first shape, wherein the second coupling portion ofthe first module is designed to engage with the first coupling portionof the tissue scaffold rack; wherein the modules fit into the troughshaped region by contacting the bottom region and substantially spanningthe two side regions; and, each of the first and second module and therack have a porous microstructure, are synthesized from polycaprolactoneand are substantially coated with calcium-deficient carbonate-containinghydroxyapatite.
 2. The biocompatible system of claim 1, comprising abioactive agent; and wherein the system comprises one or more featuresselected from the group consisting of: the first module comprises thebioactive agent the bioactive agent is present in an amount effective toinduce ossification; the bioactive agent is selected from the groupconsisting of a bone morphogenetic protein (BMP), demineralized bonematrix, a bone marrow aspirate, a transforming growth factor, afibroblast growth factor, an insulin-like growth factor, a plateletderived growth factor, a vascular endothelial growth factor, a growthand development factor-5, platelet rich plasma, and a mixture thereof;the bioactive agent is BMP2 or BMP7; and the first module and the secondmodule a first bioactive agent and a second bioactive agent,respectively, and the first bioactive agent is different than the secondbioactive agent.
 3. The biocompatible system of claim 1, furthercomprising an isolated vertebrate cell; wherein the system comprises oneor more features selected from the group consisting of: the isolatedvertebrate cell is a mammalian cell; the isolated vertebrate cell iscomprised of the first module; the isolated vertebrate cell is a stemcell; the isolated vertebrate cell is an embryonic stem cell; theisolated vertebrate cell is an adult stem cell; the isolated vertebratecell is a mesenchymal stem cell; the isolated vertebrate cell is aninduced pluripotent stem cell; and the first module and the secondmodule comprise a first isolated vertebrate cell and a second isolatedvertebrate cell, respectively, and the first isolated vertebrate cell isdifferent than the second isolated vertebrate cell.
 4. The biocompatiblesystem of claim 1 wherein each tissue scaffold module or the rackfurther comprises a ridge designed to be coupled to at least anothermodule or the rack.
 5. The biocompatible system of claim 4, wherein theridge is configured to be melted by the application of energy, andwherein the application of energy to the ridge fuses the first module orthe second module to the rack, or the first module to the second module.6. The biocompatible system of claim 1, comprising at least one pin forcoupling the tissue scaffold to tissue; and at least one featureselected from the group consisting of: (i) the tissue is bone; (ii) theat least one pin is made from a biodegradable and absorbable material;(iii) the at least one pin is for coupling to the tissue scaffold andadjacent tissue; (iv) the at least one pin is for coupling to the tissuescaffold and adjacent tissue and the coupling is accomplished by atleast one of sonically welding and applying energy to bond the at leastone pin to the tissue scaffold and the adjacent tissue; (v) the pin issynthesized from a material independently selected from a degradablepolymer and a mixture of a degradable polymer and a bioceramic; (vi) thepin is synthesized from a material independently selected from adegradable polymer and a mixture of a degradable polymer and abioceramic, and the polymer is polycaprolactone, polylactide,polyglycolide, poly(lactide-glycolide), poly(propylene fumarate),poly(caprolactone fumarate), polyethylene glycol,poly(glycolide-co-caprolactone), or mixtures thereof; (vii) the pin issynthesized from a material independently selected from a degradablepolymer and a mixture of a degradable polymer and a bioceramic, and thepolymer is polycaprolactone; (viii) the pin comprises an osteoconductivemineral coating on at least a portion of the pin; (ix) the pin comprisesan osteoconductive mineral coating on at least a portion of the pin andthe osteoconductive mineral coating is hydroxyapatite, calciumdeficientcarbonate-containing hydroxyapatite, tricalcium phosphate, amorphouscalcium phosphate, octacalcium phosphate, dicalcium phosphate, calciumphosphate, or a mixture thereof; (x) the pin comprises anosteoconductive mineral coating on at least a portion of the pin and theosteoconductive mineral coating is calcium-deficientcarbonate-containing hydroxyapatite; (xi) the pin comprises a bioactiveagent; (xii) the pin comprises a bioactive agent present in an amountthat induces ossification; (xiii) the pin comprises a bioactive agentand the bioactive agent is a bone morphogenetic protein (BMP),demineralized bone matrix, a bone marrow aspirate, a transforming growthfactor, a fibroblast growth factor, an insulin-like growth factor, aplatelet derived growth factor, a vascular endothelial growth factor, agrowth and development factor-5, platelet rich plasma, or a mixturethereof; and (xiv) the pin comprises a bioactive agent and the bioactiveagent is BMP2 or BMP7.
 7. The biocompatible system of claim 1, whereinthe first module and the second module are designed to protrude througha marrow space to provide fixation when implanted.
 8. The biocompatiblesystem of claim 7, further comprising a pin corresponding to each of thefirst module and the second module to provide additional fixation whenimplanted.
 9. The biocompatible system of claim 1, wherein the secondcoupling portion of the first module is designed to couple to the firstcoupling portion of the tissue scaffold rack via a mechanicalconnection.
 10. The biocompatible system of claim 1, wherein the firstcoupling portion is disposed at the bottom region of the tissue scaffoldrack.
 11. The biocompatible system of claim 1, wherein the firstcoupling portion is a recess and the second coupling portion is aprotuberance that is configured to fit into the recess.
 12. Thebiocompatible system of claim 1, wherein the second coupling portion isa recess and the first coupling portion is a protuberance that isconfigured to fit into the recess.
 13. The biocompatible system of claim1, wherein the system comprises a third module that is biocompatible anddegradable, wherein the third module comprises both an A connector and aB connector and is joined to the first module or the second module bythe third module A connector or B connector.